Generation of hESC reporter lines using improved gene targeting technology We propose here to develop hESC reporter lines that allow accurate real time measurement of the activity of tissue - specific promoters and that can be used for high-throughput screens and in vivo studies. To introduce reporter cassettes into hESC we will not use randomly integrating retroviral vectors but instead we will develop a method to insert reporter cassettes directly into endogenous genes. The resulting gene targeting method will not only allow production of hESC reporter lines but will also allow repair of human genetic defects in hESC without introducing potentially harmful genes, such as viral genes. Specific Aim 1: Develop methods required for gene targeting in hESCs. Methods for gene targeting in mouse ESCs are very well established, and early methods of gene targeting in hESC have been reported during the past four years. Building on this recent work we propose to systematically address each of the human ESC-specific gene targeting problems, from effective delivery of targeting constructs to efficient isolation and screening of hESC clones that retain their pluripotency. Specific Aim 2: Insert reporter cassettes into the 3'UTR of relevant endogenous genes. We propose to use the gene targeting method developed in Aim 1 to introduce fluorescent reporter constructs into the 3'UTR's of genes so their expression is driven by the endogenous promoters while preserving normal expression of the endogenous protein. We will use BAC recombineering to produce targeting vectors that have long stretches of homology, to achieve the highest possible targeting frequencies. First, we will target OCT4/POU5F1, a gene that is essential for maintenance of the undifferentiated state of hESC. Loss of a reporter signal (such as eGFP) will serve as an indication that the cells are differentiating and are no longer pluripotent;reporters can also be used to mark or select against undifferentiated cells before transplantation, or to indicate the presence of undifferentiated cells in transplants. Second, we will target NKX2-5, a cardiac-specific transcription factor that is essential for heart development and, if enough time remains, the mesoderm marker T/Brachyury and cardiac specific myosin MYH6/alpha-MHC. The resulting reporter lines will be useful in HTP screens for factors that induce effective differentiation of myocardial cells which should facilitate establishment of improved differentiation protocols, a major bottleneck in stem cell therapy. Specific Aim 3: Characterize the new hESC reporter lines. Because gene targeting requires lengthy culture under potentially harmful conditions, it is critical that the targeted lines are characterized carefully to identify potential problems before they are used in screens and assays. hESC lines developed in Aim 2 will be karyotyped and subjected to SNP genotyping and tested for pluripotency by gene expression profiling, DNA methylation profiling, in vitro differentiation and teratoma formation in mice. Public Health Relevance: This project will help improve a method to genetically alter human embryonic stem cells in order to faithfully and easily measure the activity of several heart - specific genes. This method, called gene targeting, will greatly facilitate development of protocols for differentiation of human embryonic stem cells into heart muscle cells to be used for transplantation into damaged human heart. Gene targeting works already very well for mice. It will completely avoid introduction of any viral or other foreign genes into the human cells to be used for this project and thus harbors no risk of immune rejection. Once established, it will work for any gene and can also be used for `clean ` and thus safe repair of almost any human genetic defect in stem cells and any tissue derived from stem cells.